Janos K. Lanyi

The role of Na+ in transport processes of bacterial membranes

Until recently it was generally held that transport in bacteria was linked exclusively to proton circulation, in contrast to most eucaryotic systems, which depended on Na(+) circulation. The present review is intended to trace recent developments which have led to the discarding of this idea. The discussion covers transport of Na(+) and other cations, effects of Na(+) and Na(+) gradients on metabolite transport, properties of Na(+)-dependent transport carriers, and evolutionary considerations of Na(+) transport. It is now apparent that the transport of Na(+) is an important part of energy metabolism in bacteria, and that Na(+) gradients as well as H(+) gradients are used in these systems for the conservation and transmission of energy. Two hypotheses are proposed to explain the evolution of Na/K systems, and it is presently difficult to decide between them.

Studies of the electron transport chain of extremely halophilic bacteria. I - Spectrophotometric identification of the cytochromes of halobacterium cutirubrum.

Abstract Room temperature and liquid nitrogen difference spectra of H. cutirubrum extracts show evidence of two b-type, two c-type, and one a-type cytochrome. A scheme for electron transport involving these components is suggested whereby at pH 7.2 the electron flow proceeds through cytochrome b(559), eytochrome c(555), and cytochrome a(592). At pH 9.4, however, it appears that an alternate pathway through cytochrome b(563) and cytochrome c(550) is opened. DPNH and α-glycerophosphate reduce both b-type cytochromes while succinate reduces only cytochrome b(559). After overnight incubation cytochrome b(563) is inactivated and can no longer be reduced enzymically.

Calcium transport in Halobacterium halobium envelope vesicles

Abstract Calcium transport was demonstrated in envelope vesicles prepared from Halobacterium halobium. Calcium influx is driven by a Na+ gradient (in > out), and the initial transport rates are proportional to log( Na in Na out ).The addition of monensin completely inhibits transport, but FCCP has no effect. The results suggest the existence of sodium/ calcium antiport. Calcium appears to be internally bound, since it does not exit upon the addition of monensin, although efflux of 45Ca is achieved when a 20-fold excess of calcium is added to the external medium. Calcium uptake follows Michaelis-Mententype kinetics, with a Km of 71 μ m and V of 2.0 nmol · min−1 · mg−1 of membrane protein. Illumination, which provides an electrical potential owing to light-induced proton translocation by bacteriorhodopsin, has no appreciable effect on calcium efflux from preloaded vesicles. The transport carrier requires both NaCl and KCl: Na+ for energetic reasons, required on the trans side of translocation, and K+ for regulatory reasons, required probably on the cis side of translocation. The results provide evidence for a specific sodium/calcium antiporter in these halophilic cells, similar to that found in eukaryotic cells, but unlike other investigated bacterial systems which have proton/ calcium antiporters.

Relationship between proton motive force and potassium ion transport in Halobacterium halobium envelope vesicles

Abstract Membrane vesicles prepared from Halobacterium halobium extrude protons during illumination, and a pH difference (inside alkaline) and an electrical potential (inside negative) develop. The sizes of these gradients and their relative magnitudes are dependent on a complex interaction among the proton-pumping activity of bacteriorhodopsin, Na+ extrusion through an antiport system, and the ability of K+ and Cl− to act as counterions to the electrogenic movement of H+. The net result of these variable effects is that the electrical potential is relatively independent of external pH, whereas the pH difference tends toward zero when the pH is increased to 7.5–8. Although the light-induced pH difference is greater in KCl than in NaCl, and the electrical potential smaller, this is not caused by a high permeability of the vesicle membranes to K+. The vesicle membrane is poorly permeable to K+, as shown by: lack of a K+ diffusion potential in the absence of valinomycin, light-induced electrical potentials which are in excess of the chemical potential difference for K+, and direct measurements of the slow rate of K+ influx during illumination. The finding that the rate of K+ uptake is a linear function of external K+ concentration between 0 and 1 m is inconsistent with the existence of a specific K+ permeation mechanism in these vesicles. Since at external K+ concentrations m the extrusion of Na+ during illumination proceeds much more rapidly than K+ influx, it must be concluded that the vesicles also lose Cl− and water. Measurements of light-scattering changes confirm that under these conditions the vesicles collapse. The light-induced collapse is diminished only when the inward movement of K+ is increased, either by increasing the external K+ concentration or by adding valinomycin.

Analogies between respiration and a light-driven proton pump as sources of energy for active glutamate transport in Halobacterium halobium

Abstract Cell envelope vesicles containing bacteriorhodopsin, prepared from Halobacterium halobium , have previously been shown to accumulate glutamate to high concentration gradients when illuminated. This active transport is energized by a sodium gradient (Na out + ⪢ Na in + ), which arises from Na + -efflux coupled to the light-induced H + -gradient. The oxidation of dimethyl phenylenediamine (DPD) by the vesicles also can drive uphill glutamate transport, and such transport is inhibited by KCN, azide, ionophores, or uncouplers. K T for glutamate is 1.4 × 10 −7 m under these conditions, as compared to 1.3 × 10 −7 m for light-induced transport. The respiration-induced transport of glutamate is dependent on high Na + concentrations on the vesicle exterior and requires low Na + concentrations in the interior. When Na + of increasing concentrations is included in the vesicles, transport proceeds with increasing lags, similarly to the case of light-driven transport. In vesicles to which DPD is added first, and then KCN at increasing time intervals (5 to 15 min), glutamate transport occurs after the addition of KCN, with increasing rates, even though respiration is inhibited. This indicates that the energy generated by DPD-oxidation is conserved over several minutes. These results suggest that in the case of respiration-dependent glutamate transport the translocation is also driven by a Na + -gradient; thus, there is a single glutamate transport system independent of the source of energy. The generation of such an Na + -gradient during DPD-oxidation implies that the respiration component involved, cytochrome oxidase, is functionally equivalent to bacteriorhodopsin, which acts as a proton pump.

Irregular bilayer structure in vesicles prepared from Halobactbrium cutirubrum lipids

The behaviour of the fluorescent probes, perylene and 8-anilinonaphthalene-sulfonic acid, was studied by determining fluorescence polarization in vesicles prepared from Halobacterium cutirubrum polar lipids and unfractionated lipids. In the latter case, when the non-polar lipids of this organism are included (carotenoids and squalenes, comprising 8% of the total), the environment of perylene is more fluid than in polar lipids alone. Studies of the fluorescent emission spectra of ANS and the effect of chaotropic perturbants on the motion of perylene suggest that the bilayer structure in vesicles of unfractionated lipids is distorted in such a way as to allow for the penetration of more water molecules near the hydrophobic region or to induce the probes to be nearer to the aqueous phase than is the case for the polar lipids alone. In buffers containing 100 mM MgCl2, and especially in the presence of high concentrations of NaCl as well, an irreversible thermal transition of the liquid crystalline matrix was observed in the region occupied by perylene for vesicles of unfractionated lipids. Vesicles prepared from polar lipids alone do not show such transition, and the temperature at which the transition occurs depends on the amount of non-polar lipids included. It is likely that the irregularity of the bilayer structure and the thermal breakdown are both caused by the disruptive effect of the non-polar lipids. Cell envelopes of H. cutirubrum do not show the above transition, which occurs in the lipid vesicles in ionic environments and at temperatures which are physiological for these organisms. This finding is consistent with our previous suggestion, based on spin label studies, that in H. cutirubrum the membrane proteins immobilize most or all of the lipid phase.

Studies of the electron transport chain of extremely halophilic bacteria. V. Mode of action of salts on cytochrome oxidase.

Abstract 1. Cytochrome oxidase from an extremely halophilic bacterium requires up to 5.0 M NaCl for maximal activity and stability. Differences in effectiveness among various salts in promoting enzyme activity were observed, but a minimal activity (about 30%) could be supported by all salts tested including MgCl 2 and spermine at relatively low concentrations. The enzyme activity in the presence of low concentrations of MgCl 2 was shown to be more dependent on pH but less sensitive to hydrophobic bond-breaking agents, such as ethanol and especially n -propanol, than the enzyme activity in the presence of high concentrations of NaCl. 2. Spontaneous inactivation of the enzyme in the absence of salt demonstrated that the rate of inactivation of the MgCl 2 -dependent activity has a greater temperature dependence than the rate of inactivation of the NaCl-dependent activity. The effect of temperature on the rate of inactivation of the NaCl-dependent activity was found to be greatest at neutral pH and became minimal at acid or alkaline pH. Inactivation of the enzyme in the presence of high concentrations of NaCl (by prolonged incubation) demonstrated that the enzyme was inactivated more rapidly at −10° than at 5° at pH 4.0, but not at pH 7.5. 3. These results suggest that hydrophobic forces predominate in promoting the major portion of the enzyme activity in the presence of high NaCl concentrations, while charge-shielding promotes partial enzyme activity in the presence of MgCl 2 . Hydrophobic bonds also appear to be involved in the stability of the enzyme, particularly at acid and alkaline pH, where hydrogen bonding is expected to be less extensive.

Na+ transport via Na+/H+ antiport in Halobacterium halobium envelope vesicles

Using H. halobium cell envelope vesicles containing either bacteriorhodopsin plus Na+ pump, bacteriorhodopsin alone, Na+ pump alone, or no light-responsive pigment altogether, it could be shown that the large majority of light energized Na+ extrusion in these mutants is linked to bacteriorhodopsin and to protonmotive force, and therefore must be facilitated by a Na+/H+ antiporter. Thus, the recently discovered primary Na+ pump makes only a minor contribution to light-mediated Na+ flux. The activity of the Na+/H+ antiporter appears to be independent of the presence of any photoreactive pigments, since an artifical electron donor will drive rapid Na+ extrusion in all of the vesicle preparations tested.

Passive potassium ion permeability of Halobacterium halobium cell envelope membranes.

Cell envelope vesicles, prepared from Halobacterium halobium, were loaded with 3 M KCl, suspended in 3 M NaCl, and the loss of K+ was followed at various temperatures. The Arrhenius plot of the K+-efflux rates shows a break at 30 degrees C, with higher energy of activation above the break. This temperature dependence is consistent with earlier studies of chain motions in liposomes prepared from isolated lipids. The efflux of K+ is more rapid with increasing pH between pH 5 and 7. Since these vesicles do not respire under the experimental conditions it was expected that the K+-efflux data would be related to the passive permeability of the membranes to K+. The apparent K+ permeability at 30 degrees C is 1--2 - 10(-10) cm - s-1. This value corresponds to a 5-h half-life for retained K+ in the envelope vesicles and to a probably much longer half-life in whole cells. The previously observed ability of Halobacterium to retain K+ in the absence of metabolism can thus be explained solely by the permeability characteristics of the membranes.

Studies of the electron transport chain of extremely halophilic bacteria. VIII - Respiration-dependent detergent dissolution of cell envelopes.

Abstract The kinetics of the dissolution of the cell envelope of Halobacterium cutirubrum, in the presence of cetyl trimethylammonium bromide (CTAB) or Triton X-100 as perturbing agents, indicate that respiring cells are more resistant to disruption than those inhibited with cyanide, azide, 2- heptyl-4-hydroxyquinoline -N- oxide , and phenylmercuric acetate or uncoupled with carbonylcyanide m- chlorophenylhydrazone . The difference in the rate of dissolution between respiring and inhibited cells was 2.5-fold for CTAB and 30–40-fold for Triton X-100. Although the effectiveness of CTAB in dissolving the cell envelope is increased in the presence of KCN, such inhibition does not change the uptake of the detergent by the cells.